This invention relates generally to a diagnostic aid for the removal of heparin from blood and/or plasma samples, and more particularly, to a protamine-based filter device for rapid and specific removal of heparin from blood and/or plasma samples.
Heparin, a highly sulfated mucopolysaccharide, is the most commonly used clinical anticoagulant. Its major clinical applications include, inter alia: treatment of thromboembolic patients; prophylactic treatment of high risk embolic patients; post-operative prevention of thromboembolism; and prevention of clotting and thrombus formation resulting from interventions in the circulatory system, such as cardio-vascular diagnostic procedures, catheterization, surgery of the heart and vessels, and many other procedures including extracorporeal blood circulation, such as hemodialysis, use of artificial organs and organ transplantation. Heparin is quite commonly used in medical treatment. In fact, annual production of heparin in the United States totals over 6 metric tons, or 10.sup.12 USP units, corresponding to approximately 50 million doses.
As a result of its potent anticoagulant activity, the presence of very minute amounts of heparin interferes with the accuracy of all kinetic clotting tests. Thus, it is difficult, if not impossible, to assay coagulation factors accurately. The presence of heparin also renders routine coagulation screening tests for clotting defects impossible to conduct accurately. For acutely ill, hospitalized patients receiving heparin therapy, heparin contamination in blood samples drawn through heparinized cannulae causes delays, or errors, in diagnostic evaluation of coagulation mechanism. Moreover, in plasma samples from patients being changed from heparin to coumadin therapy, heparin contamination may lead to false results indicative of resistance to coumadin therapy. Accordingly, there is present in the art a significant need for a system for neutralizing or removing heparin from blood (or plasma) samples prior to commencement of coagulation and other tests.
Conventionally, heparin is neutralized by reaction with protamine. For example, since the exact amount of heparin present in plasma samples is not known, a tedious, time-consuming protamine titration procedure is required to ensure adequate neutralization. Moreover, excess protamine or protamine-heparin complex interferes with coagulation testing and results in unpredictable testing errors. Thus, in addition to the inconvenience of this procedure, the test results are of questionable accuracy.
Another conventional approach to heparin neutralization involves the use of a microbial enzyme, heparinase, to degrade heparin. Although heparinase is currently available from Miles Scientific (Naperville, Ill.), it is extremely expensive. Additionally, heparinase is produced commercially only in small quantities for specific laboratory use and is in a relatively impure form. Thus, the use of heparinase is impractical at this point in time because large quantities of highly purified heparinase would be required for any widespread clinical use thereof.
Certain methods and procedures have been developed in an effort to remove heparin from plasma samples or to neutralize same. One known system for achieving removal employs a chromatographic technique using an ECTEOLA-cellulose (anion-exchange resin) column to remove heparin from plasma samples. However, the preparation and use of such an ion-exchange column prior to testing a plasma sample for clotting time is cumbersome, time-consuming, and inconvenient.
The known method suffers from several other serious disadvantages. In most clinical situations, only a small volume of blood is drawn from the patient for testing. However, in the known chromatographic method, a certain volume of the plasma sample is trapped in the gel matrix of the column and is not available for further use. Additionally, ECTEOLA resins lack specificity to heparin, and will disadvantageously remove other clotting factors, such as factors VIII and IX, from the plasma sample. Moreover, the method is not suitable for removing heparin from whole blood.
Other approaches to the problem of heparin removal are known in the art. One such approach employs the use of a tableted form of fibrous triethylaminoethyl cellulose (an anion-exchange resin) which is commercially available from the Organon Teknika Corporation (Durham, N.C.) under the trademark "Heparsorb." Another approach involves the formation of an insoluble protamine reaction product comprising protamine sulfate or protamine sulfate and serum albumin cross-linked with glutaraldehyde which adsorbs heparin. Although these two further approaches are more convenient than the chromatographic techniques discussed hereinabove, substantial time periods (30-50 minutes) are still required to perform the entire procedure, the steps of which include, inter alia, incubation and/or agitation of the plasma sample and heparin removal agent, and centrifugation to separate the solid reaction product with the adsorbed heparin from the plasma sample. Both of these methods require special equipment, such as a rocking aliquot mixer and a centrifuge, in order to perform the procedure properly. Moreover, triethylaminoethyl cellulose is not specific to heparin, and will thus remove other clotting factors from the plasma sample. Neither of these approaches are useful for removing heparin from whole blood. Thus, they are not suitable for preparing heparin-free blood samples for tests where whole blood is required, such as whole blood clotting time tests.
It is, therefore, an object of this invention to provide a simple, rapid and specific system for removing heparin from plasma and whole blood samples.
It is another object of this invention to provide an arrangement for removing heparin from blood products, without removing other clotting factors.
It is also an object of this invention to provide an arrangement for removing heparin from blood without impairing the ability of the blood to be tested as whole blood.
It is a further object of this invention to provide an arrangement for removing heparin from blood without requiring a large sample.
It is additionally an object of this invention to provide an arrangement for removing heparin from blood without wasting a portion of the sample.
It is yet a further object of this invention to provide an arrangement for removing heparin from blood without introducing protamine or protamine-heparin complexes into the sample.
It is also another object of this invention to provide an arrangement for removing heparin from blood without requiring the use of expensive or complex equipment.